DESCRIPTION: The applicant's research focuses on the analysis of myosin structure, assembly and function, and these efforts have led to the solution of the high resolution structure of the myosin motor domain. Models of force production based on the myosin structure postulate a bending movement between domains that is coupled to ATP hydrolysis and the interaction with actin. The interface between these domain provide the pivot fort this motion. This interface also contains a cluster of mutations associated with the disease Familial Hypertrophic Cardiomyopathy (FHC), a frequently fatal disease of striated cardiac muscle. The applicant has developed a unique approach for manipulating striated muscle myosin that is based on regulated expression of a recombinant myosin in a myogenic cell line that forms contractile myofibers. This expression system is combined with a technique for selective isolation of the recombinant protein to assay the motor activity in vitro and analyze myosin assembly and function in situ. This is a proposal to use site-directed mutagenesis to define regions of the myosin motor domain involved in conformational transitions associated with force and motion. The effect of mutations, including ones related to FHC, on the biochemical and mechanical properties of single molecules will be related to the myosin ATPase mechanism, and used to test models for the mechanism of energy transduction. The assembly and structure of myofibrils in myotubes expressing recombinant myosin will be analyzed by immunofluorescence, confocal, and electron microscopy. Disordering of the contractile apparatus is a consequence of myosin mutations associated with FHC. This outcome of the disease can be assessed only in a system where myosin assembles into contractile myofibers. In addition, molecular dissection of myosin assembly and targeting domains will be addressed by preparation of chimeric molecules containing altered rod sequences. These experiments will define the role of the rod in targeting of myosin isoforms. Finally, this project will examine the folding pathway of myosin in vitro, characterize the folding intermediates, and extend the folding analysis to the biogenesis of myosin in muscle cells. These experiments will provide critical new insights into the molecular events leading from myosin synthesis and folding to targeting, assembly and function of this crucial motor enzyme that are central to the cellular physiology of striated muscle.